高产表面活性素的重组枯草芽孢杆菌构建及培养优化

Construction and optimization of engineered Bacillus subtilis for surfactin production

摘要:

表面活性素是一种新型生物表面活性剂，因其具有良好的表面活性、可生物降解及抗菌活性，在石油开采、医药、农业和食品化妆品等领域具有广阔的应用前景。高产表面活性素菌株的获得和发酵过程优化是其商业化生产的关键。文中考察了脂肪酸合成途径对表面活性素合成的影响，强化脂肪酸生物合成关键基因以及该途径全部基因分别构建了高产表面活性素枯草芽孢杆菌Bacillus subtilis THBS-2和THBS-8，并对发酵过程中氨基酸种类及添加量、诱导剂异丙基-β-D-硫代半乳糖苷 (IPTG) 添加时间和添加量等条件对产物合成的影响进行考察，获得优化的两阶段前体添加方案：发酵3 h，加入IPTG和L-亮氨酸，使其终浓度分别为1.25 mmol/L、5 g/L；发酵24 h，添加L-亮氨酸 (终浓度5 g/L) 和浓缩培养基5 mL。优化条件下，枯草芽孢杆菌THBS-2摇瓶发酵48 h，表面活性素产量高达24 g/L；30 L发酵罐中发酵68 h，产物产量最高达到34 g/L。研究结果为表面活性素的工业化生产及应用奠定基础。

Abstract:

Surfactin has great potential applications in enhancing oil recovery, agriculture, pharmaceuticals, foods and beverages, and cosmetics due to its extraordinary surface activity, biodegradability, anti-bacterial activity and biocompatibility. Enhancing surfactin production by engineering surfactin-producer and optimizing culture conditions is the key of its industrial production and subsequent applications. In this study, the effect of fatty acid synthesis pathway on surfactin synthesis was investigated, and Bacillus subtilis THBS-2 and THBS-8 with high surfactin titer were constructed by overexpressing key genes involved in the fatty acid synthesis pathway. To optimize culture condition, the amount and adding time of isopropyl-beta-D-thiogalactopyranoside (IPTG) and amino acids were studied, and a two-stage culture method was obtained: IPTG (final concentration: 1.25 mmol/L) and leucine (final concentration: 5 g/L) were added at 3 h, leucine (final concentration 5 g/L) and condensed culture medium (5 mL) were added at 24 h. Applying this strategy, the surfactin titer of B. subtilis THBS-2 reached to 24 g/L in shake flask at 48 h and up to 34 g/L after 68 h fermentation in a 30-L fermentor. The results provide basis for large-scale production and broad application of surfactin.